Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically at the end of the specification immediately preceding the Sequence Listing and the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art.
Introduction
Ion channels are a diverse group of proteins that regulate the flow of ions across cellular membranes. In the nervous system, ion channel activity has evolved into a rapid and accurate system for intercellular communication. The electrical excitability characteristics of each neuron is in part determined by the set of channels it expresses. However, cells are also able to regulate the activity of individual channels in response to physiological or developmental events, and there is growing evidence that ion channels can be the site of integration of multiple electrical and biochemical pathways.
In vivo, ion channels appear to be multimeric proteins that are comprised of several distinct gene families, coding for channels with distinct structural and functional properties. Within a gene family, the potential for heterogeneity arising from the combinatorial assembly of different pore-forming and auxiliary subunits (Green, et al., 1995). Channel properties can be modulated by second messenger cascades and can directly bind intracellular proteins such as kinases suggesting that this may be an important way to efficiently target the signaling cascade to its effector molecule. The electrical characteristics of each neuron is, in part, determined by the set of ion channels that it expresses. However, cells are also able to regulate the activity of individual channels in response to physiological or developmental events; pore-forming (xcex1) subunits can interact with a variety of intracellular proteins, including auxiliary (xcex2) subunits, cytoskeleton-associated proteins and protein kinases (Green, et al., 1995). In addition to auxiliary (xcex2) subunits, pore-forming subunits can interact with a variety of intracellular proteins and second messenger molecules themselves including G-proteins, cytoskeleton-associated proteins and protein-kinases (Adelman, et al., 1995).
Several classes of ion channels bind directly, and are regulated by, second messenger molecules such as cyclic nucleotides (Zagotta, et al., 1996; Bruggemann, et al., 1993, and Hoshi, et al., 1995) or Ca+2 (Adelman, et al., 1992; Kohler, et al., 1996). Channels with this property may be key elements in the control of neuronal signaling, as they directly couple biochemical cascades with electrical activity. Cyclic nucleotide-gated channels (CNG) play a distinct role both in visual and olfactory signal transduction; their recent identification in the hippocampus and other regions of the brain, where cAMP and cGMP are known to mediate different forms of synaptic plasticity (Krapivinisky, et al., 1995; Frey, et al., 1993; Boshakov, et al., 1997; and Arancio, et al., 1995), suggests that CNG-channels may also contribute to the regulation of excitability in central neurons (Kingston, et al., 1996 and Bradley, et al., 1997).
The first structural gene for a K+ channel to be isolated was the gene encoded by the Shaker (Sh) locus in Drosophila melanogaster (Strong, et al., 1993; Papazian, et al., 1987). Its sequence is the prototype of a large and still expanding family of related genes (Kamb, et al., 1987; Warmke, et al., 1994). The properties of a number of well characterized K+ currents, that still await a molecular definition, predicts that other members of this family are yet to be identified (Atkinson, et al., 1991).
Although the initial members of the K+ channel superfamily were cloned by chromosomal localization of alleles responsible for functional defects (Sh, eag and slo from Drosophila; (Papazian, et al., 1987; Kamb, et al., 1987; Warmke, et al., 1991; Atkinson, et al., 1991) or following the purification of a relatively abundant protein such as the cGMP-channel from bovine retina (Liman, et al., 1994), the most widely used strategy for cloning new members of the K+ channel superfamily is by homology to these sequences. Unfortunately, this approach is not well suited for identifying more divergent sequences and potentially new branches in the phylogenetic tree of the K+ channel superfamily. Expression cloning in Xenopus oocytes can circumvent this problem; this implies a pre-existing or readily detectable physiological characterization of the channel.
The present invention provides an isolated nucleic acid molecule having sequence encoding a brain cyclic nucleotide gated (BCNG) ion channel protein or a portion thereof. The present invention additionally provides an isolated BCNG protein. The present invention further provides a composition comprising a nucleic acid, having sequence encoding a BCNG protein or a portion thereof and a carrier. The present invention further additionally provides a composition comprising a BCNG protein or portion thereof and a carrier.
In addition, the present invention provides a method of identifying a nucleic acid encoding an ion channel subunit-related protein in a sample which comprises detecting in the sample a nucleic acid molecule encoding a BCNG-related protein, positive detection indicating the presence of an ion channel subunit related protein encoding nucleic acid molecule, thereby identifying a nucleic acid encoding an ion channel subunit-related protein in the sample.
The present invention also provides a method for evaluating the ability of a compound to modulate an ion channel associated with a condition in a subject which comprises:(a) contacting a cell which expresses a BCNG-related protein in a cell culture with a compound; (b) determining the amount of BCNG-related protein expression in the cell culture; and (c) comparing the amount of BCNG-related protein expression determined in step (b) with the amount determined in the absence of the compound so as to evaluate the ability of the compound to modulate the ion channel associated with the condition in the subject.
The present invention also provides a method for evaluating the ability of a compound to interact with a BCNG-related ion channel subunit protein which comprises: (a) contacting a cell which expresses a BCNG-related protein in a cell culture with a compound under conditions permissive to formation of a complex between the compound and the BCNG-related protein; (b) determining the amount of complex formed between the compound and the BCNG-related protein in the cell culture; and (c) comparing the amount of complex formed in step (b) with the amount formed in the absence of the compound so as to evaluate the ability of the compound to interact with a BCNG-related ion channel subunit protein.
Additionally, the present invention provides a method for identifying whether a compound is capable of modulating a dysfunction in an animal comprising:(a) administering the compound to the animal under conditions permissive to formation of a complex between the compound and the BCNG-related protein; (b) determining the amount of complex formed between the compound and the BCNG-related protein in the animal; and (c) comparing the amount of complex formed in step (b) with the amount of complex formed in the animal in the absence of the compound so as to identify whether the compound is capable of modulating the dysfunction in the animal, a change in the amount of complex formed in the presence of the compound indicating that the compound is capable of modulating dysfunction in the animal.
Further, the present invention provides a method of identifying a compound which modulates the activity of BCNG-related protein which comprises: (a) introducing the nucleic acid of claim 1 into an expression system and causing the expression system to express the nucleic acid under conditions whereby an ion channel subunit protein is produced;(b) contacting the channel subunit protein with the compound; (c) determining the activity of the ion channel subunit protein; and (d) comparing the activity determined in step (c) with the activity determined in the absence of the compound, an increase or decrease in activity in the presence of the compound indicating identification of a compound which modulates the activity of BCNG-related protein.
The present invention also provides a pharmaceutical composition which comprises a compound capable of modulating BCNG-related protein activity and a pharmaceutically acceptable carrier. According to an embodiment of this invention, the carrier is a diluent, an aerosol, a topical carrier, an aqueous solution, a nonaqueous solution or a solid carrier.
Finally, the present invention provides an antibody that specifically binds to BCNG protein.